Human immunodeficiency virus (HIV), the etiological agent of acquired immunodeficiency syndrome (AIDS), enters cells by binding its envelope glycoprotein (Env) to receptor molecules and fusing its membrane with the cell membrane. Understanding how receptor molecules mediate HIV entry is critical for elucidating the mechanisms of HIV infection, and design of new antiretroviral treatments and vaccines. Following reports in 1996 that the long-sought HIV-1 fusion cofactors are chemokine receptors, we proposed that they associate physically with the HIV-1 envelope glycoprotein (Env, gp120-gp41) and the primary receptor CD4, thus serving as coreceptors (Nat. Med. 1996). According to this hypothetical model coreceptor-CD4-Env complexes are the major mediator of HIV-1 entry and can exhibit conserved conformational epitopes on the pathway to exposure of the fusion peptide that could have implications for development of vaccines and inhibitors. We also suggested that such epitopes can be identified by screening phage display libraries against the Env-CD4-coreceptor complexes. By using this approach we provided evidence that purified JRFL gp120-CD4-CCR5 complexes exhibit an epitope recognized by a novel human antibody Fab (X5) selected by screening of a phage display library from an HIV-1-infected long-term nonprogressor. The X5 epitope is inducible by CD4 and its exposure is enhanced by CCR5. X5 inhibits cell fusion mediated by envelope glycoproteins (Env) from R5, X4 and R5X4 HIV-1 isolates. These findings suggest the existence of a very early intermediate in HIV-1 entry that can serve as target for broadly neutralizing antibodies in HIV-1-infected patients, and have important implications for development of vaccines and inhibitors. Currently we continue to screen phage display libraries for identification of new entry intermediates, characterize and optimize X5 and develop new fusion proteins for use as perhaps the most potent entry inhibitors and candidate vaccines. This work is currently partially supported by award from the NIH AIDS Program. The antibody was crystallized and its structure will be known soon. The new antibody was approved for patent application by NCI. The HIV-1 envelope glycoprotein (Env, gp120-gp41) undergoes a series of conformational changes initiated by its binding to receptor molecules. I hypothesized that some of the transient Env conformations on the pathway to entry can be exhibited and retained in fusion proteins of gp120 and gp41 joined by flexible linkers. Two Env89.6 fusion proteins with linkers of 14 and 24 amino acid residues inhibited 10-100-fold more potently entry and cell fusion mediated by Envs from R5 (ADA, JRFL, SF162, Bal), X4 (HXB2, NL4-3) and R5X4 (89.6, DH12) HIV-1 isolates than a fusion protein with a 4 amino acid residue linker obtained under the same conditions, gp120-gp41 without linker or gp120. The exceptionally high inhibitory activity (50% at about 100 pM - higher than of any currently known HIV-1 entry inhibitor on molar basis) of these proteins against a variety of HIV-1 isolates, including primary isolates, may suggest the existence of broadly conserved structures that are critical for the entry process and can be exposed even in the absence of receptor-mediated activation thus opening new perspectives for elucidation of viral entry mechanisms, and development of inhibitors and vaccines. Z01 BC 10257-04